Effect of spruce PjSTS1a, PjSTS2, or PjSTS3 gene overexpression on stilbene biosynthesis in callus cultures of Vitis amurensis Rupr.

Stilbenes are natural compounds protecting plants against microbial pathogens and known to possess valuable biologically active properties. In the present study, we established transgenic grapevine callus cell cultures overexpressing three stilbene synthase (STS) genes of spruce Picea jezoensis PjSTS1a, PjSTS2, and PjSTS3. Transformation of Vitis amurensis calli with the PjSTS1a, PjSTS2, and PjSTS3 genes significantly increased total content of stilbenes in 3.6-6, 2.5-2.9, and 4.1-16.1 times, respectively, in comparison with the control calli. The most pronounced positive effect on the accumulation of stilbenes was observed for the PjSTS3-overexpressing calli where the total content of stilbenes was increased up to 3.1 mg/g DW, and the stilbene production reached 25.4 mg/L. These values were higher than those achieved for the grapevine callus cell cultures overexpressing three STS genes from V. amurensis. Thus, transformation of grapevine cell cultures with spruce STS genes with a relatively low degree of homology to the endogenous VaSTSs is a more effective strategy for induction of plant secondary metabolite biosynthesis than using the grapevine genes for the overexpression experiments.

The effect of ultraviolet-C and precursor feeding on stilbene biosynthesis in spruce Picea jezoensis.

This study examines the effects of p-coumaric (CoA) and caffeic (CaA) acids on stilbene biosynthesis in one-year-old needles of Picea jezoensis (Lindl. et Gord.) Fisch ex Carr. under control conditions and after ultraviolet (UV-C) irradiation. HPLC analysis revealed that while UV-C irradiation slightly affected the total stilbene content, CoA and CaA feeding increased the total content of stilbenes by 1.2-1.3-fold. UV-C treatment combined with CoA-feeding of the P. jezoensis cuttings exerted the most pronounced stimulatory effect on stilbene accumulation leading to the total stilbene content of 9.18 mg/g of dry weight or DW. This increase correlated with the elevated transcription of the stilbene synthase PjSTS1a and PjSTS1b genes. UV-C treatment in combination with CaA feeding of P. jezoensis did not considerably influence stilbene content. These results revealed a positive effect of UV-C radiation and phenolic precursors on the content of stilbenes in spruce needles.

Regulation of stilbene biosynthesis in plants.

MAIN CONCLUSION: This review analyzes the advances in understanding the natural signaling pathways and environmental factors regulating stilbene biosynthesis. We also discuss the studies reporting on stilbene content and repertoire in plants. Stilbenes, including the most-studied stilbene resveratrol, are a family of phenolic plant secondary metabolites that have been the subject of intensive research due to their valuable pharmaceutical effects and contribution to plant disease resistance. Understanding the natural mechanisms regulating stilbene biosynthesis in plants could be useful for both the development of new plant protection strategies and for commercial stilbene production. In this review, we focus on the environmental factors and cell signaling pathways regulating stilbene biosynthesis in plants and make a comparison with the regulation of flavonoid biosynthesis. This review also analyzes the recent data on stilbene biosynthetic genes and summarizes the available studies reporting on both stilbene content and stilbene composition in different plant families.

Stilbene biosynthesis in the needles of spruce Picea jezoensis.

Stilbenes are valuable phenolic compounds that are synthesized in plants via the phenylpropanoid pathway where stilbene synthase (STS) directly catalyzes resveratrol or pinosylvin formation. Currently, there is a lack of information about the stilbene biosynthetic pathway in spruce (Picea). Resveratrol and piceatannol derivatives have been detected in the spruce bark, needles, and roots. We analyzed seasonal variation in stilbene spectrum and content in the needles of different ages of one tree of spruce Picea jezoensis. HPLC analysis revealed the presence of nine stilbenes: t- and cis-astringin, t- and cis-piceid, t- and cis-isorhapontin, and t-piceatannol were present in amounts of 0.01-6.07 mg/g of dry weight (DW), while t-isorhapontigenin and t-resveratrol were present in traces (0.001-0.312 µg/g DW). T-astringin prevailed over other stilbenoid compounds (66-86% of all stilbenes). The highest total stilbene content was detected in one-year-old needles collected in the autumn and spring (5.4-7.77 mg/g DW). We previously cloned and sequenced full-length cDNAs of the four STS transcripts (PjSTS1a, PjSTS1b, PjSTS2, and PjSTS3) of P. jezoensis. This study presents a detailed analysis of seasonal variations in PjSTS1a, 1b, 2, and 3 transcript levels in the needles of P. jezoensis of different ages using qRT-PCR. PjSTS1a and PjSTS1b transcription was higher in the needles collected in the autumn, spring, or summer than in the winter. PjSTS2 was actively transcribed in the needles of all ages collected in the winter, spring, and summer. PjSTS3 expression did not significantly change during the year and did not depend on the age of the needles. Therefore, the data show that high levels of the stilbene glucosides and PjSTS expression are present in the needles of P. jezoensis.

Effects of 5-azacytidine-induced DNA demethylation on polyketide synthase gene expression in larvae of sea urchin Strongylocentrotus intermedius.

OBJECTIVE: To investigate the role of cytosine methylation in regulation of polyketide compounds biosynthesis in larvae of Strongylocentrotus intermedius. RESULTS: Treatment of S. intermedius larvae with 100 and 200 µM 5-azacytidine (5A) as a DNA demethylating agent significantly increased the amounts of spinochrome D and spinochrome E, as the number of pigmented cells per studied larva, in a dose-depended manner. The data on SiPks gene expression showed enhancement in 16- and 67-fold in S. intermedius larvae treated with 100 and 200 µM 5A, respectively, relative to untreated ones. Moreover, the activation of transcription factors SiGcm, SiGatae and SiKrl gene expression involved in regulation of SiPks was observed in S. intermedius larvae upon treatment with 5A, suggesting DNA methylation being powerful regulator of polyketide compounds biosynthesis. CONCLUSIONS: This is the first study to describe the role of cytosine DNA methylation in the regulation of polyketide compounds biosynthesis in sea urchins. Current study implies a negative control provided by cytosine DNA methylation machinery as a key regulator of polyketide compound biosynthesis.

[HYPOTHERMIA INFLUENCES ON OXYGEN TENSION IN THE BRAIN PARENCHYMA IN PATIENTS WITH ANEURYSMAL SUBARACHNOID HEMORRHAGE].

Aneurysmal subarachnoid hemorrhage is a serious medical and social problem. The main physiological mechanisms that determine secondary brain damage in this patients are intracranial hypertension, cerebral vasospasm, dysfunction of autoregulation mechanisms, violation of liquorodynamics and delayed cerebral ischemia. The multimodal neuromonitoring for prevention and timely correction ofsecondary brain injury factors has become routine practice in neuroICU. Measurement of oxygen tension in the brain parenchyma is one of neuromonitoring options. During the years of intensive use of this method in clinical practice the reasons for reducing the oxygen tension in the brain parenchyma were revealed, as well as developed and clinically validated algorithms for correction of such conditions. However, there are clinical situations that are difficult to interpret and even more difficult to make the right tactical and therapeutic solutions. We present the clinical observation of the patient with aneurysmal subarachnoid hemorrhage, who had dramatically reduced brain intraparenchymal oxygen pressure although prolonged hypothermia were used. Despite this, the outcome was favorable. The analysis allowed to assume that the reason for this decrease in oxygen tension in the brain parenchyma could be hypothermia itself

[Expression of SM30 (A-F) Genes Encoding Spicule Matrix Proteins in Intact and Damaged Sea Urchin Strongylocentrotus intermedius (A. Agassiz, 1863) at the Six-Arm Pluteus].

In this study we investigated expression of the SM30(A-F) gene family encoding Strongylocentrotus intermedius spicule matrix proteins during the normal and regenerative pluteus II stage (three pairs of arms). We found that SiSM30A and SiSM30B genes are expressed at high levels in the normal pluteus II sea urchin. SiSM30A is expression was also significantly upregulated in the reparative pluteus II stage 3 hours after damage. Conversely, SiSM30B was downregulated during the reparative pluteus II stage. Our findings reveal a substantial similarity between the activity of SiSM30A and SiSM30B activity in the processes of regenerative growth during the pluteus II stage and during normal development at the prism stage in Strongylocentrotus purpuratus. On the basis of our findings, we propose that normal developmental mechanisms corresponding to the preceding developmental stage are reactivated during pluteus regeneration.

RolB gene-induced production of isoflavonoids in transformed Maackia amurensis cells.

Maackia amurensis Rupr. et Maxim is a valuable leguminous tree grown in the Russian Far East, in China, and in Korea. Polyphenols from the heartwood of this species (primarily stilbenes and isoflavonoids) possess strong hepatoprotective activity. Callus culture of M. amurensis produced isoflavonoids and their derivatives. In pharmacological experiments, the callus complex was at least as effective, as the plant complex. To increase the yield of isoflavonoids, calli were transformed with the rolB gene of Agrobacterium rhizogenes. Neomycin phosphotransferase (nptII) gene was used for transgenic cell selection. Three rolB transgenic callus lines with different levels of the rolB gene expression were established. Insertion of the rolB gene caused alterations in callus structure, growth, and isoflavonoid production, and stronger alterations were observed with higher expression levels. MB1, MB2, and MB4 cultures accumulated 1.4, 1.5, and 2.1 % of dry weight (DW) isoflavonoids, respectively. In contrast, the empty vector-transformed MV culture accumulated 1.22 % DW. Isoflavonoid productivity of the obtained MB1, MB2, and MB4 cultures was equal to 117, 112, and 199 mg/L of medium, respectively, comparing to 106 mg/L for the MV culture. High level of expression of the rolB gene in MB4 culture led to a 2-fold increase in the isoflavonoid content and productivity and reliably increased dry biomass accumulation. Lower expression levels of the rolB gene in MB1 and MB2 calli did not significantly enhance biomass accumulation and isoflavonoid content, although the rolB gene activated isoflavonoid biosynthesis during the early growth stages and caused the increased content of several distinct compounds.

Age-associated alterations in the somatic mutation and DNA methylation levels in plants.

Somatic mutations of the nuclear and mitochondrial DNA and alterations in DNA methylation levels in mammals are well known to play important roles in ageing and various diseases, yet their specific contributions await further investigation. For plants, it has also been proposed that unrepaired DNA damage and DNA polymerase errors accumulate in plant cells and lead to increased somatic mutation rate and alterations in transcription, which eventually contribute to plant ageing. A number of studies also show that DNA methylation levels vary depending on the age of plant tissue and chronological age of a whole plant. Recent studies reveal that prolonged cultivation of plant cells in vitro induces single nucleotide substitutions and increases global DNA methylation level in a time-dependent fashion. Changes in DNA methylation are known to influence DNA repair and can lead to altered mutation rates, and, therefore, it is interesting to investigate both the genetic and epigenetic integrity in relationship to ageing in plants. This review will summarise and discuss the current studies investigating somatic DNA mutation and DNA methylation levels in relation to plant ageing and senescence. The analysis has shown that there still remains a lack of clarity concerning plant biological ageing and the role of the genetic and epigenetic instabilities in this process.

[Expression of the stilbene synthase genes in the needles of spruce Picea jezoensis].

Stilbenes are valuable plant phytoalexins, the biosynthesis of which is characteristic of different groups of phylogenetically unrelated plants. It is believed that all the stilbenes are the derivatives of resveratrol (3,5,4'-trihydroxy-trans-stilbene) or compounds close to it (pinosylvin or piceatannol). The last stage of the resveratrol biosynthesis takes place with the involvement of stilbene synthase or resveratrol synthase (STS). The family Pinaceae is characterized by the presence of the derivatives of pinosylvin (genus Pinus) and piceatannol (genus Picea), the biosynthetic pathways of which are scarcely examined. Previously, in different species of the genus Picea, only two stilbene synthase genes were described. On the basis of RNA isolated from the needles of spruce Picea jezoensis, the full-length cDNAs of the four stilbene synthase genes, PjSTS1a, PjSTS1b, PjSTS2, and PjSTS3, were obtained. Then, using the clone frequency analysis and real-time PCR, expression of the PjSTS1a, PjSTS1b, PjSTS2, and PjSTS3 genes was examined in the needles of P. jezoensis accessions of different age and sampled in different seasons (spring, summer, autumn, winter). Among the analyzed transcripts, the PjSTS1a and PjSTS1b genes were the most frequent, indicating their higher level of expression compared to other STS genes. The highest level of PjSTS1a and PjSTS1b expression was observed in autumn, while the level of PjSTS2 and PjSTS3 expression was the highest in spring and winter. Moreover, the highest PjSTS expression was detected in the young tissues of P. jezoensis in autumn, which may indicate a higher level of stilbene biosynthesis in these tissues.

Regulation of resveratrol production in Vitis amurensis cell cultures by calcium-dependent protein kinases.

Resveratrol is a naturally occurring plant stilbene that exhibits a wide range of valuable biological and pharmacological properties. Although the beneficial effects of trans-resveratrol to human health and plant protection against fungal pathogens are well-established, little is known about the molecular mechanisms regulating stilbene biosynthesis in plant cells. It has been recently shown that overexpression of the calcium-dependent protein kinase VaCPK20 gene considerably increased resveratrol accumulation in cell cultures of Vitis amurensis. It is possible that calcium-dependent protein kinases (CDPKs) play an important role in the regulation of resveratrol biosynthesis. In the present work, we investigated the effects of overexpression of other members of the CDPK multigene family (VaCPK9, VaCPK13, VaCPK21, and VaCPK29) on resveratrol accumulation and growth parameters of grape cell cultures. The obtained data show that overexpression of VaCPK29 increased resveratrol content 1.6-2.4-fold and fresh biomass accumulation 1.1-1.4-fold in the four independently transformed cell lines of V. amurensis compared with that in the empty vector-transformed calli. However, overexpression of the VaCPK9, VaCPK13, and VaCPK21 genes did not considerably affect resveratrol content and fresh/dry biomass accumulation in the independently transformed cell lines of V. amurensis. VaCPK29-transformed calli were capable of producing between 1.02 and 1.39 mg/l of resveratrol, while the control calli produced 0.48 to 0.79 mg/l of resveratrol. The data indicate that the VaCPK9, VaCPK13, and VaCPK21 genes are not involved in the regulation of stilbene biosynthesis in grape cells, while the VaCPK29 and VaCPK20 genes are implicated in resveratrol biosynthesis as positive regulators.

Salicylic acid induces alterations in the methylation pattern of the VaSTS1, VaSTS2, and VaSTS10 genes in Vitis amurensis Rupr. cell cultures.

KEY MESSAGE: Salicylic acid (SA) treatment selectively reduced the cytosine DNA methylation of stilbene synthase ( STS ) genes and stimulated resveratrol production in cell cultures of Vitis amurensis. The effect of salicylic acid (SA) on plant growth, flowering time, and fruit number is known to correlate with the level of DNA methylation, while the potential correlation between SA-induced changes in DNA methylation and biosynthesis of secondary metabolites has not been studied. Trans-resveratrol, a naturally occurring plant phenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In this study, cell cultures of Vitis amurensis capable of producing t-resveratrol were used as a model system to study whether the SA-induced increase in t-resveratrol production is associated with changes in DNA methylation of stilbene synthase (STS) genes. T-resveratrol is synthesized via the phenylpropanoid pathway, in which STS genes are the key enzymes. Treatment of V. amurensis callus cultures with SA significantly increased t-resveratrol production and the expression of certain STS genes (e.g., VaSTS2 and VaSTS10). A marked decrease in the methylation of the VaSTS2 and VaSTS10 genes in response to SA was demonstrated using bisulfite sequencing, while no considerable changes were detected in the methylation of VaSTS1, a constitutively and highly expressed STS gene. The obtained results show that SA treatment selectively reduced cytosine methylation of VaSTS genes. The data suggest that selective DNA demethylation of particular STS genes could be necessary for the activation of t-resveratrol biosynthesis in response to SA. This finding provides an insight into the mechanism of SA action and biosynthesis of secondary metabolites in plant cells.

The methylation status of plant genomic DNA influences PCR efficiency.

During the polymerase chain reaction (PCR), which is a versatile and widely used method, certain DNA sequences are rapidly amplified through thermocycling. Although there are numerous protocols of PCR optimization for different applications, little is known about the effect of DNA modifications, such as DNA methylation, on PCR efficiency. Recent studies show that cytosine methylation alters DNA mechanical properties and suggest that DNA methylation may directly or indirectly influence the effectiveness of DNA amplification during PCR. In the present study, using plant DNA, we found that highly methylated plant DNA genomic regions were amplified with lower efficiencies compared to that for the regions methylated at a lower level. The correlation was observed when amplifying stilbene synthase (STS1, STS10) genes of Vitis amurensis, the Actin2 gene of Arabidopsis thaliana, the internal transcribed spacer (AtITS), and tRNAPro of A. thaliana. The level of DNA methylation within the analyzed DNA regions has been analyzed with bisulfite sequencing. The obtained data show that efficient PCRs of highly methylated plant DNA regions can be hampered. Proteinase K treatment of the plant DNA prior to PCR and using HotTaq DNA polymerase improved amplification of the highly methylated plant DNA regions. We suggest that increased DNA denaturation temperatures of the highly methylated DNA and contamination with DNA-binding proteins contribute to the hampered PCR amplification of highly methylated DNA. The data show that it is necessary to use current DNA purification protocols and commercial kits with caution to ensure appropriate PCR product yield and prevent bias toward unmethylated DNA amplification in PCRs.

VaCPK20 gene overexpression significantly increased resveratrol content and expression of stilbene synthase genes in cell cultures of Vitis amurensis Rupr.

Resveratrol, a naturally occurring plant phenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In the present investigation, we show that transformation of a Vitis amurensis Rupr. cell suspension with the gene VaCPK20 for a calcium-dependent protein kinase (CDPK) under the control of double CaMV 35S promoter increased resveratrol production in five independently transformed cell lines in 9-68 times compared with control cells. The VaCPK20-transformed calli were capable of producing 0.04-0.42 % dry wt. of resveratrol, while the control calli produced up to 0.008 % dry wt. of resveratrol Also, we characterized expression of stilbene synthase (STS) genes in the five VaCPK20-transgenic cell lines of V. amurensis. In all VaCPK20-transgenic cell lines, expression of VaSTS7 increased; while expression of VaSTS1 decreased. We suggest that transformation of V. amurensis calli with the VaCPK20 gene induced resveratrol accumulation via enhancement of expression of the VaSTS7 gene involved in resveratrol biosynthesis. The obtained data first demonstrate that overexpression of a CDPK gene resulted in increased accumulation of a stilbenoid phytoalexine in transgenic plant cells. We propose that the VaCPK20 gene could play an important role in the regulation of resveratrol biosynthesis in grape cells.

[Regulation of somatic embryogenesis in Panax ginseng C. A. Meyer cell cultures by PgCDPK2DS1].

We isolated the full-length cDNA of PgCDPK2DS1 gene, the expression of which was significantly increased at early stages of embryo development in cell cultures of ginseng P. ginseng 2c3. Interest in this gene also was supported by its nonstandard structure: the amino acid sequence of the PgCDPK2DS1 gene contained only the N-terminal domain and 80% of the kinase domain. Overexpression of the PgCDPK2DS1 gene in nonembryonic calli 1c resulted in the appearance of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture 1c-2d. Also, expression of the plant embryogenesis marker genes WUS and SERK significantly increased in cell culture 1c-2d. The observed embryo-like structures were at early stages of embryo development; attempts to obtain an adult plant from these embryo-like structures were unsuccessful. Overexpression of PgCDPK2DS1 gene in the embryonic cell culture PG resulted in a decrease of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture PG-2d. Moreover, expression of the plant embryogenesis marker genes WUS and SERK and expression of the endogenous PgCDPK2DS1 significantly decreased in the cell culture PG-2d. Thus, for the first time it was shown that the PgCDPK2DS1 gene is involved in the regulation of somatic embryogenesis in P. ginseng cell cultures.

[Effect of plant stilbene precursors on the biosynthesis of resveratrol in Vitis amurensis Rupr. cell cultures].

The biosynthesis of resveratrol after the application of a precursor for biosynthesis, i.e., phenylalanine (Phe), has been studied. The application of Phe has been shown to increase significantly the expression of the phenylalanine-ammonia-lyase (PAL) and stilbene synthase (STS) genes and enhance the production of resveratrol by 8.5 times. Data on resveratrol production after the addition of Phe and coumaric acid (CA) were compared with known analogs.

Differences in the methylation patterns of the VaSTS1 and VaSTS10 genes of Vitis amurensis Rupr.

Resveratrol is a plant-derived phenol but the mechanism that regulates its biosynthesis remains unidentified. Stilbene synthase (STS) catalyzes resveratrol formation in vivo and we have proposed that inducers of resveratrol production affect STS expression through an unidentified epigenetic mechanism. To investigate the role of DNA methylation in resveratrol biosynthesis, we treated both rolB transgenic and empty vector control Vitis amurensis cell cultures with the DNA demethylation agent, 5-azacytidine. Treated cells had increased resveratrol production through activation of VaSTS10 expression. The lowest levels of cytosine methylation were at the 5'- and 3'-ends of the VaSTS1 protein-coding sequence. Cytosine methylation decreased mostly at the 5'- and 3'-ends of VaSTS10 after azaC treatment with an intriguing regularity in the number of cytosine nucleotides within the 5'- and 3'- ends of the protein-coding sequences. Thus, cytosine methylation is crucial for the regulation of the resveratrol biosynthetic pathway.

The role of canonical and noncanonical pre-mRNA splicing in plant stress responses.

Plants are sessile organisms capable of adapting to various environmental constraints, such as high or low temperatures, drought, soil salinity, or pathogen attack. To survive the unfavorable conditions, plants actively employ pre-mRNA splicing as a mechanism to regulate expression of stress-responsive genes and reprogram intracellular regulatory networks. There is a growing evidence that various stresses strongly affect the frequency and diversity of alternative splicing events in the stress-responsive genes and lead to an increased accumulation of mRNAs containing premature stop codons, which in turn have an impact on plant stress response. A number of studies revealed that some mRNAs involved in plant stress response are spliced counter to the traditional conception of alternative splicing. Such noncanonical mRNA splicing events include trans-splicing, intraexonic deletions, or variations affecting multiple exons and often require short direct repeats to occur. The noncanonical alternative splicing, along with common splicing events, targets the spliced transcripts to degradation through nonsense-mediated mRNA decay or leads to translation of truncated proteins. Investigation of the diversity, biological consequences, and mechanisms of the canonical and noncanonical alternative splicing events will help one to identify those transcripts which are promising for using in genetic engineering and selection of stress-tolerant plants.

Structure and expression profiling of a novel calcium-dependent protein kinase gene, CDPK3a, in leaves, stems, grapes, and cell cultures of wild-growing grapevine Vitis amurensis Rupr.

KEY MESSAGE : VaCDPK3a is actively expressed in leaves, stems, inflorescences, and berries of Vitis amurensis and may act as a positive growth regulator, but is not involved in the regulation of resveratrol biosynthesis. Calcium-dependent protein kinases (CDPKs) are known to play important roles in plant development and defense against biotic and abiotic stresses. It has previously been shown that CDPK3a is the predominant CDPK transcript in cell cultures of wild-growing grapevine Vitis amurensis Rupr., which is known to possess high resistance against environmental stresses and to produce resveratrol, a polyphenol with valuable pharmacological effects. In this study, we aimed to define the full cDNA sequence of VaCDPK3a and analyze its organ-specific expression, responses to plant hormones, temperature stress and exogenous NaCl, and the effects of VaCDPK3a overexpression on biomass accumulation and resveratrol content in V. amurensis calli. VaCDPK3a was actively expressed in all analyzed V. amurensis organs and tissues and was not transcriptionally regulated by salt and temperature stresses. The highest VaCDPK3a expression was detected in young leaves and the lowest in stems. A reduction in the VaCDPK3a expression correlated with a lower rate of biomass accumulation and higher resveratrol content in calli of V. amurensis under different growth conditions. Overexpression of the VaCDPK3a gene in the V. amurensis calli significantly increased cell growth for a short period of time but did not have an effect on resveratrol production. Further subculturing of the transformed calli resulted in cell death and a decrease in expression of the endogenous VaCDPK3a. The data suggest that while VaCDPK3a acts as a positive regulator of V. amurensis cell growth, it is not involved in the signaling pathway regulating resveratrol biosynthesis and resistance to salt and temperature stresses.